DNA methyltransferases are a family of enzymes that promote the covalent addition of a methyl group to a specific nucleotide base in a molecule of DNA. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor. Four active DNA methyltransferases have been identified in mammals. They are named DNMT1, DNMT2, DNMT3A and DNMT3B.
DNMT1 is the most abundant DNA methyltransferase in mammalian cells and considered to be the key maintenance methyltransferase in mammals. It predominantly methylates hemimethylated CpG di-nucleotides in the mammalian genome and is responsible for maintaining methylation patterns established in development. The enzyme is about 1620 amino acids long, the first 1100 amino acids constituting the regulatory domain, and the remaining residues constituting the catalytic domain. These are joined by Gly-Lys repeats. Both domains are required for the catalytic function of DNMT1. DNMT3 is a family of DNA methyltransferases that can methylate hemimethylated and unmethylated CpG at the same rate. The architecture of DNMT3 enzymes is similar to DNMT1 with a regulatory region attached to a catalytic domain.
Recent work has revealed how DNA methylation and chromatin structure are linked at the molecular level and how methylation anomalies play a direct causal role in tumorigenesis and genetic disease. Much new information has also come to light regarding DNA methyltransferases, in terms of their role in mammalian development and the types of proteins they are known to interact with. Rather than enzymes that act in isolation to copy methylation patterns after replication, the types of interactions discovered thus far indicate that DNA methyltransferases may be components of larger complexes actively involved in transcriptional control and chromatin structure modulation. These findings should enhance the understanding of the myriad roles of DNA methylation in disease, as well as leading to novel therapies for preventing or repairing these defects.
Small molecule DNA methyltransferase inhibitors are well documented in the art and include, for example, decitabine, azacitabine, zebularine, procainamide, procaine, hydralazine, ((−)-epigallocatechin-3-gallate (EGCG) and RG108.
Decitabine or 5-aza-2′-deoxycytidine (trade name Dacogen) is the compound 4-amino-1-(2-deoxy-b-D-eryth ro-pentofuranosyl)-1,3,5-triazin-2(1H)-one.
Azacitidine (trade name Vidaza) is the compound 4-amino-1-β-D-ribofuranosyl-s-triazin-2(1H)-one, the structure of which is shown below.

Azacitidine is an anti-neoplastic pyrimidine nucleoside analog used to treat several subtypes of myelodysplastic syndrome, diseases caused by abnormalities in the blood-forming cells of the bone marrow which result in underproduction of healthy blood cells. The drug exerts a cytotoxic effect on rapidly dividing cells, including cancerous cells, and may help restore normal function to genes controlling proper cellular differentiation and proliferation.
Azacitidine is specifically indicated for the treatment of the following myelodysplastic syndrome subtypes: refractory anemia, refractory anemia with ringed sideroblasts (if accompanied by neutropenia or thrombocytopenia or requiring transfusions), refractory anemia with excess blasts, refractory anemia with excess blasts in transformation and chronic myelomonocytic leukemia.
Azacitidine is believed to exert its antineoplastic effects by causing hypomethylation of DNA and direct cytotoxicity on abnormal haematopoietic cells in the bone marrow. The concentration of azacitidine required for maximum inhibition of DNA methylation in vitro does not cause major suppression of DNA synthesis. Hypomethylation may restore function to genes that are critical for differentiation or proliferation. The cytotoxic effects of azacitidine cause the death of rapidly dividing cells, including cancer cells that are no longer responsive to normal growth control mechanisms. Non-proliferating cells are relatively insensitive to azacitidine.
Another known DNA methyltransferase inhibitor is zebularine, also known as 1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one or 2-pyrimidone-1-β-D-riboside, the structure of which is shown below.

Other known DNA methyltransferase inhibitors are non-nucleoside analogues, for example, procainamide, procaine, hydralazine and ((−)-epigallocatechin-3-gallate (EGCG).
Procainamide (trade names Pronestyl, Procan, Procanbid) is the compound 4-amino-N-(2-diethylaminoethyl)benzamide, the structure of which is shown below.

Procainamide has been shown to inhibit DNA methyltransferase activity and reactivate silenced gene expression in cancer cells by reversing CpG island hypermethylation. Procainamide specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1), the mammalian enzyme thought to be responsible for maintaining DNA methylation patterns during replication.
Procaine is the compound 2-(diethylamino)ethyl-4-aminobenzoate, the structure of which is shown below.

Procaine is a DNA-demethylating agent that is understood to inhibit DNA methyltransferases by interfering with enzyme activity.
Hydralazine (Apresoline) is the compound 1-hydrazinophthalazine monohydrochloride, the structure of which is shown below.

((−)-Epigallocatechin-3-gallate (EGCG) is a catechin analogue having the structure shown below.

EGCG is understood to inhibit DNMT activity and reactivate methylation-silenced genes in cancer cells.
Another known DNA methyltransferase inhibitor is RG108, also known as N-phthalyl-1-tryptophan, the structure of which is shown below.

RG108 is a DNA methyltransferase inhibitor that is understood to inhibit DNA methyltransferases by interfering with enzyme activity. In particular, RG108 is believed to reactivate tumor suppressor gene expression (p16, SFRP1, secreted frizzled related protein-1, and TIMP-3) in tumor cells by DNA demethylation. RG108 also inhibits human tumor cell line (HCT116, NALM-6) proliferation and increases doubling time in culture.
It is well established in the art that active pharmaceutical agents can often be given in combination in order to optimise the treatment regime.
Qin T et al (2007, 13, Clin. Cancer Res. 4225-4232) disclose the effect of combinations of cytarabine and decitabine in various human leukemic cell lines. Likewise, Kong X B et al (1991, Molecular Pharmacol. 39, 250-257) suggest that 5-azacitidine causes upregulation of dCK in a cell line that is resistant to cytarabine, resulting in a decrease in the IC50 value for cytarabine from 12.5 to 0.55 μM.
Combinations of DNA methyltransferase inhibitors and 1-(2-C-cyano-2-dioxy-β-D-arabino-pentofuranosyl)-N4-palmitoyl cytosine (also known as “CYC682” or sapacitabine), or a metabolite thereof, are described in WO 2009/150405 (Cyclacel Limited). Pharmaceutical compositions comprising such combinations, and their use in treating various proliferative disorders are also described in WO 2009/150405.
The present invention seeks to provide a new dosing regimen for known pharmaceutical agents that is particularly suitable for the treatment of proliferative disorders, especially acute myeloid leukemia (AML). More specifically, the invention centres on the surprising and unexpected effects associated with using certain pharmaceutical agents in combination.